The present invention relates to a sensitizer for tumor treatment, which is to be used in the treatment of such a disease as tumor by ultrasound irradiation, and to an ultrasonic treatment system and a modality of ultrasonic therapy, in which that sensitizer is used. More specifically, it relates to a sensitizer given the amphiphilicity (both hydrophilicity and lipophilicity) and thus increased in its accumulation in the tumor tissue as derived from a sensitizer having a xanthene dye structure by modifying that structure, and to an ultrasonic treatment system and a modality of ultrasonic therapy, in which that sensitizer is used.
In recent years, even in cancer treatment, attention has been paid to the quality of life of posttreatment patients and, therefore, treatment modalities by which the region of tumor alone might be treated selectively and which would cause less damage to the body except for the region of tumor have been demanded. To develop such a modality of therapy, sonodynamic therapy which uses ultrasound and a sensitizer therefor (Reference 1: Jpn. J. Cancer Res., 80, 219-222, 1989) has become a target of study.
In this modality, an agent administered in advance is activated locally as an antitumor agent by means of waves and thereby the tumor region alone is treated, like in photodynamic therapy (Reference 2: Cancer Res., 39, 146-151, 1979) which is already in clinical application.
In photodynamic therapy, there is a theoretical problem that since the wavelength of a laser beam, thereof the attenuation coefficient in the living body, is restricted, the target of treatment is limited to superficial tumor located at most several millimeters deep from the surface. On the other hand, in sonodynamic therapy, the relation between the wavelength of an ultrasonic wave and the attenuation coefficient thereof is appropriate to the human body and therefore the therapy is characterized in that ultrasonic waves can be focused not only on superficial tumor but also on tumor located in a deep region. Therefore, sonodynamic therapy, if put to practical use, is expected to play a role in low invasive therapy, along with photodynamic therapy, in the target of therapy where it is preferable.
Referring to the above-mentioned sonodynamic therapy, various techniques have been explored through physical and chemical approaches and new techniques have been developed with respect to both system and agent.
In sonodynamic therapy, acoustic cavitation is thought to play an important role in the mechanisms of that therapy. Acoustic cavitation is a phenomenon of bubbles formed upon ultrasound irradiation growing and collapsing, and biological reactions are produced mechanically and chemically by the very high pressure and temperature temporarily generated on the occasion of the collapse.
Techniques for efficiently introducing this acoustic cavitation have been developed, for example the technique comprising irradiating ultrasound while switching the acoustic fields (Reference 3: JP Kokai H02-126848) and the technique comprising superimposing the second harmonics on the fundamentals (Reference 4: International laid-open Patent Specification WO 94/06380). When these techniques are used, acoustic cavitation can be introduced at a lower level of acoustic intensity.
On the other hand, through the agent side approach, the present inventors previously proposed an acoustic cavitation promoter for lowering the acoustic intensity threshold required for introducing acoustic cavitation in which a sensitizer having a xanthene dye structure is used (Reference 5: International laid-open Patent Specification WO 98/01131).
Further, it is known, as a basic mechanism of sonodynamic tumor treatment, that the use of a substance capable of generating active oxygen due to a chemical effect of ultrasound can increase the sonochemical antitumor effect (Reference 6: JP Kokai H06-29196). It is also known that sensitizers having a xanthene dye structure, which are acoustic cavitation promoters, are active oxygen-producing substances.
In addition to the foregoing, it is important that the agent be capable of being accumulated in tumor tissues. In sonodynamic therapy, the spatial selectivity of the region to be treated is attained principally by the irradiation of focused ultrasound. However, when the treatment of that tumor located in a deep region of a living body or the treatment of a tumor species showing a complicated boundary between normal tissues and tumor tissues, such as infiltrative or disseminated tumor, is taken into consideration, it is essential for realizing safer and more effective therapy that the agent to be administered be itself capable of being accumulated in tumor tissues.
Therefore, for an agent to be ideal for sonodynamic therapy, it is desired that the agent have three characteristics simultaneously, namely (1) acoustic cavitation promoting activity, (2) ability to produce antitumor effects and (3) potential for being accumulated in tumor tissues.
Tumor tissues tend to take up lipoproteins to maintain their vigorous growth and have lymphoid tissues poorly formed, hence lipoproteins once taken up by them can hardly be excreted therefrom. A highly lipophilic agent, which has high affinity for lipoproteins, is thought to migrate with lipoproteins and be readily accumulated in tumor tissues. On the other hand, for an agent to be administered to the living body, it is essential that the agent have hydrophilicity as well.
Therefore, for an agent to be capable of being accumulated in tumor cells, it is desirable that the agent be endowed with a balance between lipophilicity and hydrophilicity and be high in both characteristics, namely be amphiphilic. Sensitizers having a xanthene dye structure are generally very high in hydrophilicity but very low in lipophilicity and, therefore, for improving their accumulation in tumor tissues, it is conceivable that their lipophilicity be increased. For increasing the lipophilicity, it is a common practice to introduce a lipophilic functional group such as an alkyl group. With such type of compound, however, the hydrophilic region is remote from the lipophilic region in the molecule, hence the compound will supposedly take a micelle-like form in body fluids; the possibility that the above characteristics (1) and (2) originally possessed by the compound be adversely affected is high.
Accordingly, it is an object of the present invention to provide a sensitizer containing a xanthene dye structure and endowed with the characteristic (3), namely ability to be accumulated in tumor tissues, without the above characteristics (1) and (2) being impaired.
One of the approaches for obtaining the compound aimed at by the invention is the one previously proposed by the present inventors and described in the above-cited reference 5. This comprises dimerizing a sensitizer having a xanthene dye structure and serving as an acoustic cavitation promoter simultaneously having antitumor activity to thereby render the same lipophilic.
Since, however, such sensitizer dimerization has problems on synthesizes, for example a lot of labor is required for isolation, the inventors made attempts to obtain a desired compound through another approach. The process in which the present invention was created is described below in detail.
When an attempt is made to improve the lipophilicity of an agent by mere introduction of a lipophilic group, in particular when a lipophilic group is introduced while sacrificing a hydrophilic functional group, the hydrophilicity may possibly decrease and the administration to a living body may become difficult. It is conceivable that a hydrophilic group be newly introduced to avoid the decrease in hydrophilicity due to lipophilic group introduction. However, in such a case, too, if the lipophilic group and hydrophilic group introduced are intramolecularly remote from each other, the product agent will become micelle-like in body fluids, as discussed above, and the cavitation promoting effect will expectedly decrease due to such change in characteristics.
From such viewpoint, the inventors made investigations concerning derivatives having a lipophilic group (R1) and a hydrophilic group (R2) introduced on the same carbon atom, as shown below by Formula 1, as candidate sensitizers containing a xanthene dye structure. 
In Formula 1, X and Y each is a halogen atom or a hydrogen atom, Z is an alkali metal atom, such as Na or K, or a hydrogen atom, R1 is a lipophilic group, R2 is a hydrophilic group and R3 may be a lipophilic group R1 or a hydrophilic group R2 or a hydrogen atom.
During the investigations, it was revealed that, for the effect of the hydrophilic group R2 to be produced to the full, it is important to employ, as the lipophilic group R1, a straight chain alkyl group, for instance, which can take various three-dimensional configurations according to the milieu (e.g. solvent).
The number of carbon atoms contained in the straight chain alkyl group is desirably 3 to 30 and the optimal number of carbon atoms may vary depending on the level of hydrophilicity of the hydrophilic group R2 and/or the structure of the lipophilic group R1.
It was further found that when an alkyl group having a side chain or chains is employed in lieu of such straight chain alkyl group to serve as the lipophilic group R1, the same effect can be produced. Further, it was found that a functional group derived from a straight or side-chain-containing alkyl group by substitution of a double bond and/or a triple bond for part of the single bonds (Cxe2x80x94C) thereof, a functional group derived from a straight or side-chain-containing alkyl group by substitution of an aromatic ring (for example benzene ring), or a functional group derived from a straight or side-chain-containing alkyl group by partial introduction of an ether linkage, ester linkage, amido group, sulfur atom or the like is also effective in producing the same effect. Specific examples of the lipophilic group R1 are shown in Table 1. They are by no means limitative of the scope of the invention, however.
In Table 1, there are shown straight alkyl groups, side-chain-containing alkyl groups, functional groups derived from a straight alkyl group by substitution of a double bond for part of the single bonds (Cxe2x80x94C) thereof, functional groups derived from a straight alkyl group by substitution of a triple bond for part of the single bonds (Cxe2x80x94C) thereof, functional groups resulting from the binding of a phenyl group to the terminus of a straight alkyl group, functional groups resulting from the binding of phenyl isothiocyanate to the terminus of a straight alkyl group, functional groups resulting from the binding of phenylsulfonyl chloride to the terminus of a straight alkyl group, straight chain ether, straight chain ester and straight chain amido groups. The use of one of these is preferred in the practice of the present invention.
Desirable as the compound to be used in the practice of the invention are sensitizers containing a xanthene dye structure such as those having the structure represented by Formula 1, for example rose bengal (x=Cl, Y=I), phloxine B (X=Cl, Y=Br), 3,4,5,6-tetrachlorofluorescein (X=Cl, Y=H), erythrosine B (X=H, Y=I), and the sodium salt of each of them.
It is known that when sensitizers having such a xanthene dye structure have intramolecularly a halogen atom or a functional group capable of chemically binding to a thiol group or amino group included in proteins in the cell membrane, an improvement in their effect can result. In the practice of the present invention as well, the effect can be increased by employing such a structure.
It is desirable that the halogen comprise at least one of fluorine (F), chlorine (Cl), bromine (Br) and iodine (I). It is particularly desirable that one of X and Y in Formula 1 be a halogen.
The functional group capable of binding to proteins desirably comprises at least one of aziridine, amido, isothiocyanate, imido and sulfonyl chloride groups. In Formula 1, the carbon atom (C) to which the lipophilic group R1 and hydrophilic group R2 are bound is bonded to a sensitizer having a xanthene dye structure via ester bonding. It is to be understood that the scope of the invention is not limited by such mode of binding. In the case of compounds having such a structure, however, the introduction by ester bonding is the most expedient.
As a method of obtaining compounds to be used in the practice of the invention, a method is shown below for synthesizing compounds by introducing a straight alkyl group as lipophilic group R1 and a carboxyl group as hydrophilic group R2 into a sensitizer having a xanthene dye structure (such compounds, namely -xcex1-carboxyalkyl esters, are hereinafter referred to as xe2x80x94COOHxe2x80x94Cn, n being the number of carbon atoms contained in the straight alkyl group). The method is by no means limitative of the scope of the invention, however.
These compounds can be synthesized according to the route shown in Equation 1 with reference to Reference 7 (Z. Naturforsch., B; Anorg. Chem. Org. Chem., 39B, 474-484, 1984) and the desired compounds containing a xanthene dye structure as represented by Formula 4 are obtained. 
In Equation 1, X and Y are as defined above referring to Formula 1 and n represents the number of carbon atoms in the alkyl group and desirably n=3 to 20.
This reaction is the so-called nucleophilic replacement reaction between the carboxylate part (COO) of the sensitizer having a xanthene dye structure as represented by Formula 2 and the halogenide of a fatty acid as represented by Formula 3. The particulars of this synthesis are described later in Example 1.
Variations of the number of carbon atoms in the lipophilic alkyl group shown in Formula 4 can be obtained by submitting fatty acid halogenides differing in the number of carbon atoms to the reaction. The halogenides of this kind are commercially available.
The above halogenides can also be synthesized from a fatty acid having the desired number of carbon atoms by bromination of the a position thereof using phosphorus trichloride as a catalyst as described in Reference 8 (Org. Synth., I, 115-116, 1941).
In the practice of the invention, the hydrophilic group R2 desirably comprises at least one of a carboxy group or its soluble salt, a sulfonic acid group or its soluble salt, a sulfuric acid ester group or its soluble salt, a hydroxy group or its soluble salt, an amine group or its soluble salt, a quaternary ammonium group or its soluble salt and a phosphoric acid group or its soluble salt.
In some instances, the group R2 may contain lipophilic structures such as alkyl group, ether linkage, amido group, ester linkage or benzene ring or a like. For example, it is known that an ether linkage has low hydrophilicity itself, but an ether linkage can form polyethylene glycol resulting from the combination of a plurality of ether bonds, alkyl groups and hydroxy groups, thereby polyethylene glycol shows high hydrophilicity. Examples of the functional groups or soluble salts mentioned above as well as examples of the combinations thereof are shown in Table 2, without any meaning restrictive of the scope of the invention.
In Table 2, R represents a functional group such as a chain-like alkyl group, and a cyclic N compound such as a pyridinium salt is preferred as the quaternary ammonium salt. As for n, it is preferred that n=3 to 20.
While Equation 1 illustrate a synthetic pathway for xe2x80x94COOHxe2x80x94Cn compounds in which the hydrophilic group R2 is a carboxy group, compounds having another hydrophilic group introduced can be synthesized in the same manner using a halogenide having the respective hydrophilic group.
For example, the halogenide of a compound in which the hydrophilic group is a sulfonate can be synthesized by the method described in Reference 9 (J. Am. Chem. Soc., 62, 1044, 1940). This synthetic pathway is illustrated by Equation 2 and Equation 3.
In Equation 3, X and Y are as defined above referring to Formula 1 and n represents the number of carbon atoms in the alkyl group and preferably n=10 to 30.
First, an unsymmetrically dihalogenated alkane (Formula 5) is sulfonated using a sulfite (Formula 6). The sulfonated compound (Formula 7) obtained is reacted with a xanthene dye, whereby the desired sulfonated compound containing a xanthene dye structure as represented by Formula 8 is obtained. The particulars of this synthesis are shown later in Example 2.
Compounds in which the hydrophilic group R2 is a carboxylate can be prepared from xe2x80x94COOHxe2x80x94Cn by converting the carboxylic acid to the carboxylate (this method is mentioned in detail in Example 3).
To sum up, the sensitizer for sonodynamic therapy according to the invention which contains a xanthene dye structure is characterized by its structure such that at least one hydrophilic group R2 and at least one lipophilic group R1 are always bound to one and the same carbon atom. This lipophilic group R1 desirably comprises a straight alkyl group containing 3 to 30 carbon atoms, and the alkyl group may partly contain at least one of a branched alkyl group, double bond, triple bond, aromatic ring (e.g. benzene ring), ether linkage, ester linkage, amido group, sulfur, etc.
The hydrophilic group R2 desirably comprises at least one of a carboxy group or its soluble salt, a sulfonic acid group or its soluble salt, a sulfuric acid ester group or its soluble salt, a hydroxy group or its soluble salt, an amine group or its soluble salt, a quaternary ammonium group or its soluble salt and a phosphoric acid group or its soluble salt.
The ultrasonic treatment system according to the invention comprises a transducer for targeting, an ultrasonic therapeutic transducer, a controller and an acoustic-cavitation monitor for detecting the acoustic cavitation induced by the sensitizer of the invention and adequately maintaining the site and intensity of ultrasound irradiation, among others.
The acoustic cavitation detecting means is not restricted but may be any means capable of measuring a physical phenomenon caused by acoustic cavitation. Thus, means for measuring such an acoustic phenomenon as a subharmonic or harmonic waves or means for measuring light emission may be used.
In the following, the constitution of the present invention is described.
The sensitizer (A) for tumor treatment according to the invention is characterized in that it has the skeleton of xanthene dyes and has a structure such that at least one hydrophilic group and at least one lipophilic group are bound to one and the same carbon atom occurring in a functional group bound to a carbon atom in that skeleton. It is a sensitizer for tumor treatment which can enhance the effect of tumor treatment as produced by ultrasound irradiation.
The sensitizer for tumor treatment according to the invention has a functional group capable of chemically binding to the thiol or amino group and the functional group comprises at least one of aziridine, amido, isothiocyanate, imido and sulfonyl chloride groups.
More specifically, the sensitizer (B) for tumor treatment according to the invention is characterized in that it has the skeleton of xanthene dyes and has a structure such that at least one hydrophilic group and at least one lipophilic group are bound to one and the same carbon atom occurring in a functional group bound to a carbon atom in that skeleton and that it has the structure represented by Formula 1. In Formula 1, X and Y each is a halogen atom or a hydrogen atom, Z is an alkali metal atom or a hydrogen atom, R1 is a lipophilic group, R2 is a hydrophilic group and R3 is a hydrophilic group R1 or a hydrophilic group R2 or a hydrogen atom. Typical examples of this sensitizer for tumor treatment are described in the following.
X and Y each is a halogen atom and Z is an alkali metal atom.
The lipophilic group R1 is a lipophilic group comprising a straight alkyl group containing 3 to 30 carbon atoms.
The lipophilic group R1 is one of straight alkyl groups, side-chain-containing alkyl groups, functional groups derived from a straight or side-chain-containing alkyl group by substitution of a double bond for part of the single bonds thereof, functional groups derived from a straight or side-chain-containing alkyl group by substitution of a triple bond for part of the single bonds thereof, functional groups derived from a straight or side-chain-containing alkyl group by the binding of an aromatic ring thereto, and functional groups derived from a straight or side-chain-containing alkyl group by partial introduction of at least one of an ether linkage, an ester linkage, an amido group and a sulfur atom.
The hydrophilic group R2 is a hydrophilic group comprising at least one of a carboxy group or its soluble salt, a sulfonic acid group or its soluble salt, a sulfuric acid ester group or its soluble salt, a hydroxy group or its soluble salt, an amine group or its soluble salt, a quaternary ammonium group or its soluble salt and a phosphoric acid group or its soluble salt.
The ultrasonic treatment system according to the invention comprises an ultrasonic imaging probe for irradiating a target for treatment in a living body with ultrasound for imaging to thereby obtain a diagnostic ultrasonic image of the target for treatment, an ultrasonic therapeutic transducer for irradiating the target for treatment with ultrasound for treatment, a controller for adjusting the intensity and/or focus of the ultrasound for treatment based on the diagnostic ultrasonic image, an ultrasonic transducer for generating bubbles which generates ultrasound for irradiating the target for treatment with administered sensitizer (A) for tumor treatment illustrated hereinabove and generates bubbles, which are stabilized by the sensitizer for tumor treatment, by irradiation with the ultrasound, and a display device for displaying the diagnostic ultrasonic image obtained by the ultrasonic imaging probe and contrasted by the bubbles. After the diagnostic ultrasonic image as contrasted by the bubbles is displayed on the display device, the ultrasound for treatment is irradiated on the target for treatment.
In another aspect, the ultrasonic treatment system according to the invention comprises an ultrasonic imaging probe for irradiating a target for treatment in a living body with ultrasound for imaging to thereby obtain a diagnostic ultrasonic image of the target for treatment, an ultrasonic therapeutic transducer for irradiating the target for treatment with ultrasound for treatment, a controller for adjusting the intensity and/or focus of the ultrasound for treatment based on the diagnostic ultrasonic image, an ultrasonic transducer for generating bubbles which generates ultrasound for irradiating the target for treatment with administered sensitizer (B) for tumor treatment illustrated hereinabove and generates bubbles, which are stabilized by the sensitizer for tumor treatment, by irradiation with the ultrasound, and a display device for displaying the diagnostic ultrasonic image obtained by the ultrasonic imaging probe and contrasted by the bubbles. After the diagnostic ultrasonic image as contrasted by the bubbles is displayed on the display device, the ultrasound for treatment is irradiated on the target for treatment.
The modality of ultrasonic therapy according to the present invention comprises (1) the step of irradiating a target for treatment in a living body with the ultrasound for imaging as generated from an ultrasonic imaging probe to thereby obtain a diagnostic ultrasonic image of the target for treatment, (2) the step of adjusting the intensity and/or focus of the ultrasound for treatment as generated from an ultrasonic therapeutic transducer based on the diagnostic ultrasonic image, (3) the step of irradiating the target for treatment with administered sensitizer (A) for tumor treatment illustrated hereinabove with the ultrasound generated by an ultrasonic transducer for generating bubbles to thereby generate bubbles, which are stabilized by the sensitizer for tumor treatment, by ultrasound irradiation, (4) the step of displaying the diagnostic ultrasonic image obtained by the ultrasonic imaging probe and contrasted by the bubbles and (5) the step of irradiating the target for treatment with the ultrasound for treatment after displaying of the diagnostic ultrasonic image as contrasted by the bubbles.
In another aspect, the modality of ultrasonic therapy according to the invention comprises (1) the step of irradiating a target for treatment in a living body with the ultrasound for imaging as generated from an ultrasonic imaging probe to thereby obtain a diagnostic ultrasonic image of the target for treatment, (2) the step of adjusting the intensity and/or focus of the ultrasound for treatment as generated from an ultrasonic therapeutic transducer based on the diagnostic ultrasonic image, (3) the step of irradiating the target for treatment with administered sensitizer (B) for tumor treatment illustrated hereinabove with the ultrasound generated by an ultrasonic transducer for generating bubbles to thereby generate bubbles, which are stabilized by the sensitizer for tumor treatment, by ultrasound irradiation, (4) the step of displaying the diagnostic ultrasonic image obtained by the ultrasonic imaging probe and contrasted by the bubbles and (5) the step of irradiating the target for treatment with the ultrasound for treatment after displaying of the diagnostic ultrasonic image as contrasted by the bubbles.
The sensitizer for ultrasonic therapy according to the invention possesses both lipophilicity and hydrophilicity. It is important that the lipophilic group and hydrophilic group are bound to one and the same carbon atom in the molecule. When they are remote from each other, no sufficient effect is obtained. The sensitizer of the invention is an agent having the function of inducing acoustic cavitation and the function of revealing an antitumor effect and capable of being highly accumulated in tumor tissues. Namely, the sensitizer of the invention is high in selectivity for tumors and can enhance the effect of tumor treatment, thus enabling the treatment of malignant tumor with ultrasound.